Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication Is Independent of Anaphase-Promoting Complex Activity

Abstract

The anaphase-promoting complex, or cyclosome (APC/C), is a large E3 ubiquitin ligase composed of 14 subunits. The activity of APC/C oscillates during the cell cycle to ensure a timely transition through each phase by promoting the degradation of important cell cycle regulators. Of the human herpesviruses, cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) both impair the activity of APC/C during their lytic replication cycle through virus-encoded protein kinases. Here, we addressed whether the oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) deregulates the activity of APC/C during the lytic replication cycle. To this end, we used the well-characterized iSLK.219 cell model of KSHV infection and established a new infection model of primary lymphatic endothelial cells (LECs) infected with a lytically replicating KSHV BAC16 mutant. In contrast to those of EBV and HCMV, the KSHV lytic cycle occurs while the APC/C is active. Moreover, interfering with the activity of APC/C did not lead to major changes in the production of infectious virus. We further investigated whether rereplication stress induced by the unscheduled activation of the APC/C-CDH1 complex affects the number and integrity of KSHV viral episomes. Deep sequencing of the viral episomes and host chromosomes in iSLK.219 cells revealed that, while distinct regions in the cellular chromosomes were severely affected by rereplication stress, the integrity of the viral episomes remained unaltered. IMPORTANCE DNA viruses have evolved complex strategies to gain control over the cell cycle. Several of them target APC/C, a key cellular machinery that controls the timely progression of the cell cycle, by either blocking or enhancing its activity. Here, we investigated the activity of APC/C during the lytic replication cycle of KSHV and found that, in contrast to that of KSHV's close relatives EBV and HCMV, KSHV lytic replication occurs while the APC/C is active. Perturbing APC/C activity by depleting a core protein or the adaptor proteins of the catalytic domain, and hence interfering with normal cell-cycle progression, did not affect virus replication. This suggests that KSHV has evolved to replicate independently of the activity of APC/C and in various cell cycle conditions.

Parthenolide inhibits proliferation of cells infected with Kaposi’s sarcoma-associated herpesvirus by suppression of the NF-κB signaling pathway

“…iSLK.219 is a rKSHV.219 infected iSLK cell line. The iSLK.219 cell is the well model cells for screening the antiviral agents against KSHV and the mechanism study [23,35]. For the SK-RG cells, based on the AIDS patients are susceptible to KSHV, and some patients have neurological symptoms.…”

Investigation of key miRNAs and Target-mRNA in Kaposi's sarcoma using bioinformatic methods

“…First, we extracted total RNA from cells using the Trizol method [ 18 ]; The RNA was then transcribed into cDNA using the Reverse Transcription Kit (RR047A, TaKaRa), and the cDNA was amplified using Universal Fluorescence Quantitative PCR Kit (BL697A, Biosharp). The qRT-PCR was performed under the following cycling parameters: 50 °C for 2 min, 95 °C for 10 min; 40 cycles at 95 °C for 15 s and 60 °C for 1 min; followed by melt-curve parameters: 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s. The RNA transcript level was presented as relative quantification (RQ) using the comparative cycle threshold (ΔΔCT) method.…”

Seroprevalence and Biomarkers Detection of Human Herpes Virus (HHV-8) in Patients with Kaposi Sarcoma

“…HHV8 shows no clear cytolyticimpactat the lysis phase and thus, molecular methods are required for its detection (such asamplication of PCR for specific genes like latency factor LANA, or immunostaining of surface antigens K8.1) [14], Human umbilical vein endothelial cells (HUVEC) and the BAC36 strain were used to provide a highly effective cellular model of infection and viral lytic replication. Such a model allows for defining the lytic-latent phase, revealing that viral production begins at 1-2 days and continues to 10-15 days after infections, followed by its entering latent states as episomes bound with cellular DNA-associated histones where it can remain for the complete lifespan of hosts [15]. HHV8 causes considerable changes in cellular biochemistry and physiology during latent infection, involving changes in HHV8 permeability of cells, resistance to toxic medicines, increased resistance to stressful conditions, enhancement of glycolysis as well as higher expressions to the insulin receptor (IR) [16].…”